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Advances in NSCLC: histologic distinction between adenocarcinoma and squamous cell carcinoma

By Mark D. Cross

Lung cancer causes 28% of all cancer mortality. In the United States, 226,160 new cases of this disease and 160,340 deaths are expected in 2012, accounting for 14% of cancer diagnoses, according to American Cancer Society estimates.¹ That makes lung cancer the deadliest form of cancer in the U.S. Worldwide, there were 1.6 million cases of lung cancer and 1.3 million deaths in 2008.²

Several histotypes of lung cancer exist, including small cell lung cancer, which accounts for 14% of all lung cancers.¹ In this article, we will discuss differentiation and therapy options for the most common histotype, non-small cell lung cancer (NSCLC), which constitutes approximately 85% of lung cancers.^1,3,4 For patients with advanced NSCLC, estimated survival is six months⁴ while overall survival at one year was 43% in 2003 to 2006.¹ Five year survival is estimated at 16% since the vast majority of patients are diagnosed with advanced disease.¹ Five year survival rises dramatically to 52% if the disease is diagnosed when it is still localized, but only 15% of patients are diagnosed at this stage,¹ underscoring the need for superior diagnostic tests for early detection.

The two main subtypes of NSCLC are squamous cell carcinoma (SqCC), which arises from epithelial cells, and adenocarcinoma (LADC), which arises from glandular epithelial tissue and is frequently seen in patients who have never smoked tobacco. Until recently, a diagnosis of NSCLC was sufficient to provide guidance for therapy,^3,5 since treatment was similar for both SqCC and LADC. However, with the advent of powerful new medications that are contraindicated and may be fatal for certain subtypes of NSCLC, accurate differentiation of these subtypes is imperative for medication selection. As a result, the recent International Multidisciplinary Classification of Lung Adenocarcinoma addresses the importance of differentiating LADC from SqCC.⁶

Clinical requirements for new targeted drugs

With the introduction of therapeutic agents that are indicated specifically for non-squamous NSCLC, differentiation of NSCLC subtypes has become critically important. Determination of squamous cell carcinoma versus adenocarcinoma is important to optimize the likelihood of patient response to Avastin® (bevacizumab)⁷ and Alimta® (pemetrexed),⁸ a folate analog. Treatment of SqCC patients with either drug is contraindicated, and, in the case of Avastin, has been associated with pulmonary hemoptysis (severe hemorrhage) and a significant mortality rate.⁷

Avastin (bevacizumab) is a Vascular Endothelial Growth Factor (VEGF) inhibitor that is indicated for non-squamous NSCLC patients and is designed to inhibit cellular signaling that initiates angiogenesis (blood vessel formation). Tumors have high metabolic needs to support tumor growth, so are dependent on increased vascularization to supply nutrients and oxygen needed for growth. Tissues automatically produce VEGF in response to hypoxia (reduced oxygen),⁹ which occurs in a tumor as it outgrows its vascular supply channels. Also, tumors may produce excess VEGF, which causes angiogenesis when this protein binds to a specific receptor (VEGF Receptor 2 or VEGFR2) on nearby blood vessels. Blockage of VEGF from binding to its receptor by Avastin prevents the angiogenic action of VEGF, starving the tumor (Figure 1).

Figure 1. Avastin blocks VEGF signaling

VEGF binds to VEGF receptor (VEGFR2) on endothelial cells.⁹ As a result, VEGFR2 forms dimers and sends signals through several pathways which each prepare the cell in different ways to mount an angiogenic response. Avastin (bevacizumab) inhibits this process by binding competitively to the VEGF binding site.

However, Avastin is contraindicated in squamous cell carcinoma patients. Research demonstrates that this medication can cause a significant mortality rate⁷ in squamous cell carcinoma patients through hemoptysis, or severe hemorrhaging of the lung, perhaps caused by the formation of leaky or unstable blood vessels.

A third type of therapy which is dependent on lung tumor histology is the class of EGFR inhibitors. EGFR mutations cause constitutive (permanently turned on) EGFR signaling, resulting in uncontrolled tumor growth, increased angiogenesis, and failure of apoptotic (cell death) pathways which are designed to kill tumor cells&emdash;all favoring tumor metastasis. EGFR mutations sensitize NSCLC to EGFR inhibitors such as gefitinib (Iressa®)and erlotinib (Tarceva®),¹⁰ since these drugs are designed to inhibit EGFR pathway signaling. The majority of EGFR mutations are found in lung adenocarcinoma,³ therefore reinforcing the need for accurate identification of NSCLC subtype.

Many other proteins are the target of oncology medications in NSCLC clinical trials. These include HER2, present in 2% of NSCLCs, primarily never-smokers, East Asian populations, and women.¹¹ Trastuzumab (Herceptin®) showed response rates of 21% to 40% in one trial.¹² MET proto-oncogene has been shown to be overexpressed in 40% of lung cancers¹¹ and MET gene amplification has been shown to lead to EGFR inhibitor resistance. Several medications aimed at inhibiting MET are currently in development.¹¹ While survival and clinical improvements are limited with the current drug armamentarium, these and new compounds offer hope to patients with this destructive disease.

Challenges in NSCLC differentiation

Lung cancer was the most commonly misdiagnosed cancer worldwide in 2008.¹¹ Although NSCLC differentiation can be made based on hematoxylin and eosin (H&E) staining in many cases, this is more difficult with advanced disease, which typically has poorly differentiated tumors. Features that assist with morphological differentiation, such as keratinization or gland formation, are not always distinguishable, particularly when minimal tissue is available as in biopsies. Adding to this difficulty, 70% of diagnoses are made from small samples such as fine-needle aspiration (FNA) or transbronchial biopsy specimens,³ Thus tissue, and the number of tests that may be performed using the tissue, are extremely limited, particularly when molecular testing must be performed.

Concordance rates between pathologists in differentiating NSCLC have been shown to be as low as 81% based on H&E alone, which may be insufficient as a basis for therapy.^13,14

In order to meet this challenge, immunohistochemistry (IHC) has been shown to be a valuable adjunct to H&E staining, particularly for poorly differentiated tumors.³ To make more effective use of limited tissue, multiplex IHC approaches have been developed, wherein two or more antibodies directed against morphologically distinct antigens are added to the same tissue sample.^15,16 Each antibody can be detected using a different color chromogen.

Sensitivity and specificity of adenocarcinoma vs. SqCC differentiation has typically been fairly low, even with use of immunohistochemistry testing. One study reported accurate classification in 30 of 39 (77%) poorly differentiated NSCLC cases.¹⁷ Another study reported 74.6 % sensitivity using antibodies TTF-1 and p63.¹⁸ Clearly, in the light of the urgency of accurate differentiation of NSCLC, increased confidence in these diagnoses would be clinically helpful.

More sensitive and specific markers

Fortunately, the emergence of new biomarkers has enabled increased sensitivity and specificity for this critical distinction using immunohistochemistry (IHC). Three such markers are Napsin A, Desmoglein 3 and p40. These, in combination with some of the more traditional markers such as TTF-1, CK5 and p63, may enable much more confident identification of SqCC and LADC.

Napsin A is a pepsin-like aspartic proteinase and functions to mature pulmonary surfactant proteins.¹⁹ It is expressed in type II pneumocytes and in adenocarcinomas of the lung and kidney³ and has been demonstrated to be a more sensitive and specific marker of adenocarcinoma than TTF-1, with 74% to 87% sensitivity and specificity approaching 100% reported.^19,20 In addition to expression in LADC, TTF-1 is also expressed in thyroid (80%) and small cell carcinoma of the lung, which may mimic NSCLC especially when poorly differentiated. Napsin A is expressed in 57% of renal tumors, so a combination of Napsin A and TTF-1 is helpful if metastatic tumors are part of the differential.²⁰

Whithaus et al³ report that "The sensitivity and specificity of different clones can profoundly affect the diagnosis," specifically referring to clone choice for Napsin A and TTF-1. They continue: "TTF-1 and Napsin A are a dynamic pair that can help rule out small cell carcinoma (TTF-1 +, Napsin -) and rule in ACA simultaneously (TTF-1+, Napsin -)."

Desmoglein 3 is an extracellular adhesion protein of the cadherin superfamily that is found in desmosomes, which are cell-cell junctions found in epithelial, myocardial, and other cell types. So this marker is highly specific for primary lung SqCC with sensitivity and specificity of 83% and 100% respectively.

p63 is commonly used as an aid in differentiation of lung adenocarcinoma vs. SqCC. While this marker offers sensitivity approaching 100% for SqCC, its utility is limited because p63 is expressed in from 16% to 65% of LADC^21,22 as well as the majority of large cell lymphomas, both of which can mimic NSCLC. p63 consists of several isoforms (variants) such as ΔNp63 and TAp63, which differ both in the structure of the N-terminal domain and function (Table 1). Antibodies for p63 such as clone 4A4, commonly used today in clinical laboratories, recognize both the ΔNp63 and the TAp63 isoforms.²¹

Table 1. p40 antibody recognizes a different epitope than p63 antibody, which confers increased specificity for Lung SqCC.

Figure 2. p40 antibody staining lung squamous cell carcinoma.

The p40 antibody recognizes the ΔNp63, but not the TAp63 isoform of p63 (Table 1). Since the ΔNp63 isoform is the predominant form of p63 in basal and progenitor cells and ΔNp63 functions as a stem cell factor and an oncogene,²¹ the p40 antibody which recognizes ΔNp63 has been reported to offer equivalent sensitivity to p63, but with greatly increased specificity. In a study performed at Memorial Sloan Kettering and Johns Hopkins hospitals, LADC, SqCC and large cell lymphoma samples were tested for p40 and p63 expression by IHC. Large cell lymphoma was included in the study since it can "present as a solitary thoracic mass, and its epithelioid morphology may closely mimic non-small cell carcinoma."²¹ Both antibodies were expressed in 100% of SqCC tumors (n = 470).²¹ However, p63 was expressed in 31% of LADC samples and 54% of large cell lymphomas (sensitivity 100%, specificity 60%), whereas p40 was expressed in only 3% of LADC (focal staining only) and none of the large cell lymphomas, giving a sensitivity of 100% and specificity of 98% for this critical differentiation. In another study of 200 NSCLC cases, p63 (4A4) was positive in 18% of LADC, whereas p40 was negative in all LADC cases²¹ while both antibodies stained 100% of SqCC cases.²² Bishop et al²¹ write:

In summary, p40 is equivalent to p63 in sensitivity for squamous cell carcinoma, but is markedly superior to p63 in specificity, which eliminates a potential pitfall of misinterpreting a p63-positive adenocarcinoma or unsuspected lymphoma as squamous cell carcinoma. These findings strongly support the routine use of p40 in place of p63 for the diagnosis of pulmonary squamous cell carcinoma.²¹

Several studies have examined the sensitivity and specificity of multiple antibody panels to identify SqCC and LADC. Ring et al,¹⁸ guided by genomic studies and a selection process that included 105 protein markers, developed a novel five antibody immunohistochemistry panel targeting the proteins TRIM29, CEACAM5, SLC7A5, MUC1, and CK5/6 that were combined into one test using a weighted algorithm. This test, Pulmotype, was validated in three independent cohorts of more than 1100 samples. They report sensitivity of 88.6% and NPV of 84.8% vs. 74.6% and 63.7% respectively using an immunohistochemistry panel of TTF-1 and p63. Pulmotype also showed a 50% reduction in unclassifiable samples and equivalent specificity compared to the TTF-1 and p63 panel. The Pulmotype test, available from Clarient, complements "the morphologic assessment already performed by pathologists when sufficient specimen is available, or by replacing morphologic assessment when the specimen is too sparse for morphologic interpretation" (clarientinc.com).

Tacha et al¹⁶ developed a pulmonary panel using immunohistochemistry methods that used a combination of traditional markers such as TTF-1 and p63 and newer, highly sensitive and specific markers such as napsin A and desmoglein 3 to aid in identification of lung SqCC and adenocarcinoma. Antibody selection was guided by the sensitivities and specificities of the individual antibodies. A protocol was developed that used a sequential series of antibodies such that 85% of cases would only require one or two immunohistochemistry slides for complete analysis, while providing a follow on sequence of IHC tests for more difficult cases.

This pulmonary panel of antibodies resulted in a reported sensitivity of 93% with 100% specificity for identification of NSCLC squamous cell carcinoma and adenocarcinoma. Two new antibodies specifically developed and optimized for the diagnosis of LADC and lung SqCC, a rabbit polyclonal Napsin A and a mouse monoclonal Desmoglein 3 [BC11], were the key elements of the antibody panel. The authors comment, "Given the increased difficulty of diagnosing poorly differentiated tumors, the ability of this 6-antibody panel to classify 96% and 87% of moderately and poorly differentiated cases, respectively, is of particular value, especially when limited tissue for molecular testing is an issue."

Accurate differentiation of lung SqCC and LADC is imperative, and IHC can provide important adjunctive tests to assist oncologists in making appropriate treatment decisions for each patient's histotype. New tools and markers have become available in reference laboratory and IVD kit formats respectively that provide additional data with which to make these clinical decisions.

Table 1. Total mislabeled specimens

Avastin® and Alimta® are registered trademarks of Genentech and Eli Lilly respectively.

References

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Mark D. Cross is Senior Director of Sales and Marketing for California-based Biocare Medical.

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